This proposal is aimed at further developing and applying a general method that allows one to systematically add amino acids with novel physical, chemical and biological properties to the genetic codes of both prokaryotic and eukaryotic organisms. This methodology involves the generation of orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that uniquely recognize a noncoding codon and do not cross react with any of the endogenous tRNAs and aminoacyl-tRNA synthetases in the host organism. The amino acid specificity of the aminoacyl-tRNA synthetase is then modified such that it aminoacylates its cognate tRNA with only the desired unnatural amino acid and no endogenous amino acid. Using this method, a large number of structurally diverse unnatural amino acids have been incorporated efficiently and with high fidelity in response to nonsense and frameshift codons in bacteria, yeast and mammalian cells. We now propose to extend these studies in several directions. The first involves the evolution of tRNA-aaRS pairs in eukaryotes and/or prokaryotes specific for a series of biophysical probes/tools including fluorescent and photocaged amino acids, and amino acids that can be used to conformationally constrain the protein backbone. Another effort will focus on technical improvements to increase suppression efficiencies, protein yields and the number of orthogonal tRNA-aaRS pairs. The final direction will involve novel biomedical applications of this methodology including the generation of vaccines with enhanced immunogenicity, and the synthesis of novel antibody conjugates for imaging and therapeutic applications. PUBLIC HEALTH RELEVANCE: The ability to genetically encode unnatural amino acids beyond the common twenty will significantly enhance our ability to manipulate protein structure. The result will be new tools to study protein function in vitro and in vivo. It should also be possible to create homogenous "chemically mutated" therapeutic proteins and vaccines with enhanced activities and improved pharmacology.